This invention relates to the fixation of hybrid vigor and other traits through apomixis (asexual seed formation) in flowering plants (angiosperms). More particularly, it provides methods for “stabilizing” apomixis in natural or man-made facultative apomicts (plants capable of sexual and apomictic reproduction) such that sexually-derived progeny, which are occasionally produced facultatively from such apomictic plants, tend to be apomictic like the mother plant, though otherwise genetically recombined, instead of being sexual revertants. It also provides methods for “controlling” apomixis, in natural or synthetic apomicts, such that such apomicts express obligate apomixis (no capacity for sexual seed formation), obligate apomixis except when induced to be facultatively apomictic, or facultative apomixis except when induced to be obligately apomictic. This invention uses genetic, cytogenetic, and molecular modifications to prevent genetic recombination among loci critical to the expression of apomixis (stabilization of apomixis) and controls the percentage of seeds that are derived apomictically by controlling frequency of sexually-derived seeds in natural or synthetic facultative apomicts (control of apomixis).
The types of apomixis referred to in the present patent application cause asexual seed formation. Accordingly, seeds of apomictic plants contain embryos that are genetic clones of the mother plant. Such forms of apomixis comprise adventitious embryony and gametophytic apomixis (referred to hereinafter as apomixis), which is commonly divided into apospory and diplospory. S. E. Asker & L. Jerling, Apomixis in Plants (CRC Press 1992) (hereinafter, “Asker & Jerling”).
Developmental signals responsible for apomixis preempt megasporogenesis by inducing precocious embryo sac formation from either the megaspore mother cell (MMC) (diplospory) or from somatic nucellar cells (apospory). Fertilization is also preempted by precocious embryony, which often occurs before the stigma is receptive to pollen. Wobble in the intensity of signals responsible for apomixis allows for the facultative expression of sexual reproduction within apomictic plants. Hence, in most apomicts, a certain percentage of seeds produced by a single apomictic plant will form sexually, and this percentage is often influenced by environmental factors. Asker & Jerling. In Antennaria-type diplospory, signals for precocious embryo sac formation occur very early, completely preventing meiosis. In Taraxacum-type diplospory, signals for embryo sac formation are less precocious and affect the MMC after meiosis has initiated. In Hieracium-type apospory, nucellar cells are affected by the precocious and ectopic embryo-sac-inducing signals, and the affected somatic nucellar cells undergo three rounds of endomitosis to produce an 8-nucleate embryo sac. In Panicum-type apospory, only two rounds of endomitosis occur, resulting in mature 4-nucleate embryo sacs. In adventitious embryony, embryos form from cells other than the egg, including cells of the nucellus, integument(s), synergids, and antipodals. Asker & Jerling.
Technologies that induce, stabilize, and control the expression of apomixis in crops have the potential of revolutionizing plant breeding and becoming essential to competitive agribusiness worldwide. With such systems, breeders will “clone” highly desirable plants (exhibiting hybrid vigor, transgenic traits, and the like) through the plant's own seed—generation after generation. Yield increases resulting from the fixation of hybrid vigor of inbred crops such as wheat (15%) and rice (35%) will be economically exploited on a large scale for the first time, which will make apomixis of immense commercial value worldwide. Because cloning occurs through seed, apomixis may become the most cost effective plant mechanism for transferring biotechnological and productivity advances to marginal farmland in the developed world and to resource poor farmers in developing nations. Apomixis may become among the most valuable genetic tools for plant breeders in the 21st century. At a recent conference on apomixis, the following conclusion was reached: “The prospect of introducing apomixis into sexual crops presents opportunities so revolutionary as to justify a sustained international scientific effort. If apomixis were generated with a sufficiently high degree of flexibility, the impact on agriculture could be profound in nature and extremely broad in scope.” The Bellagio Apomixis Conference, Why is Apomixis Important to Agriculture (1998).
Four modes of inheritance for apomixis have been proposed during the past 100 years: chromosomal non-homologies (wide hybridization), quantitative inheritance, simple inheritance, and complex inheritance. The chromosomal nonhomology hypothesis, championed by A. Ernst, Bastardierung als Ursache der Apogamie im Pflanzenreich (Fischer, Jena 1918), states that apomixis is a function of chromosomal nonhomology and is one of several cytogenetic anomalies caused by wide hybridization. According to this theory apomixis is not controlled by genes directly, but is a consequence of divergence in chromosome structure. This hypothesis is no longer considered valid mainly because apomixis occurs in plants whose chromosomes appear to be homologous. J. G. Carman, Asynchronous Expression of Duplicate Genes in Angiosperms May Cause Apomixis, Bispory, Tetraspory, and Polyembryony, 61 Biol. J. Linnean Soc. 51-94(1997).
The quantitative-mode-of-inheritance hypothesis is also considered to be invalid. In the mid 20th century, it was supported by Muntzing, who believed apomixis resulted from a delicate balance of few to many recessive genes, and Powers, who believed that recessive genes caused the three major components of apomixis: failure of meiosis, apomictic embryo sac formation, and parthenogenesis. Asker & Jerling.
During the past 40 years, most apomixis scientists, including Bashaw, Nogler, Savidan, Sherwood, and Harlan, have supported the simple inheritance hypothesis, i.e. that one or two dominant genes confer apomixis. Asker & Jerling. This conclusion initially appears well founded in that Mendelian analyses repeatedly produce simple inheritance segregation ratios, e.g. 1:1 apomictic to sexual progeny ratios are often produced in crosses made between sexual and apomictic plants. Y. Savidan, Apomixis: Genetics and Breeding, 18 Plant Breed. Rev. 13-86 (2000). However, despite years of effort, no apomixis gene has been identified or isolated.
In the late 1990s, the duplicate-gene asynchrony hypothesis or hybridization-derived floral asynchrony theory (hereinafter, “HFA theory”) was proposed for the evolution of apomixis. J. G. Carman, 61 Biol. J. Linnean Soc. 51-94 (1997). It implies complex inheritance and is based on a synthesis of concepts from various fields of biology. According to this hypothesis, the mode of inheritance for apomixis is not simple; nor is it simply quantitative, at least not in the standard way of viewing quantitative inheritance. In contrast, it is complex and is best explained through a series of five tenets, which build upon each other. The first three tenets have been published, J. G. Carman, 61 Biol. J. Linnean Soc. 51-94 (1997), and are summarized below. The last two tenets comprise unpublished concepts novel to the present invention and are presented herein.
First, apomixis is a developmentally-disjunct hybrid phenotype. Apomixis is disjunct from, not intermediate to, its parental female reproductive phenotypes, which, for convenience, are labeled parental phenotypes A and B. Plants exhibiting phenotypes A or B undergo normal sexual reproduction. Phenotypic differences between A and B are detected cytoembryologically through state-of-the-art microscopy techniques. They are not casually observed, which is why they have not been described previously.
Second, parental phenotypes A and B are distinctly different from each other with regard to the time periods in which meiosis, embryo sac formation, and embryony occur relative to gross ovule development.
Third, parental phenotypes A and B are themselves quantitatively inherited. Hence, nearly obligate apomixis, where most ovules of a given plant produce functional apomictic embryo sacs, is expressed because of polygenic heterozygosity. In populations of agamic complexes (populations of interbreeding sexual and apomictic species), multiple alleles exist for many of the critical loci, i.e. the critical loci are polymorphic. The polygenic heterozygosity responsible for nearly obligate apomixis involves specifically divergent alleles, which are maintained in natural populations because of natural selection. In contrast, facultative apomixis, where sexual and apomictic seeds commonly develop on the same plant, occurs when some of the more critical loci required for obligate apomixis become homozygous (or acquire alleles that encode similar schedules of ovule development) through genetic segregation.
Based on the HFA theory, efficient procedures for synthesizing facultatively apomictic plants from sexual plants have been described. J. G. Carman, Methods for Producing Apomictic Plants, WO 98/33374 (1998) (hereby incorporated by reference). These methods are used to produce highly apomictic plants that may or may not be genetically stable as defined above. The solution offered in WO 98/33374 is to produce highly apomictic plants, i.e. to reduce, as far as possible, the occurrence of sexual seed formation in apomictic hybrids by identifying or producing (through breeding) pairs of parent lines that are appropriately divergent in their female reproductive schedules such that facultative sexual development is minimized in the facultatively apomictic hybrid progeny. Synthetic apomicts produced in this manner may be used as apomictic hybrid lines for several to many generations before the harvested seed becomes useless for replanting due to serious contamination from seeds of sexual revertants. The contaminating revertant seeds are products of genetic segregation, and their presence degrades agronomic value. This situation would be analogous to the mixing of inferior F2 and later generations of seed with elite F1 hybrid seed in a conventional hybrid seed production program. The result would be an agronomically inferior product. WO 98/33374 did not address the subject of stabilization and control of apomixis. Hence, methods for modifying an apomict once it is synthesized were not provided.
In view of the above, it would be advantageous to provide methods that permit development of apomictic lines that are obligate, obligate unless induced to be facultative, or facultative unless induced to be obligate. By inducing facultative apomixis, the apomictic line can be improved, by conventional breeding strategies, and subsequently returned to the obligately apomictic condition for many years of field production. It should be appreciated that these and other advantages of the present application (discussed below) represent major advancements in the state-of-the-art.